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Supplementary Information to

DNA or Protein? Capillary Zone Electrophoresis–Mass Spectrometry

Rapidly Elucidates Metallodrug Binding Selectivity

Christian Artner,a,b,c Hannah U. Holtkamp,c Wolfgang Kandioller,a Christian G. Hartinger,c

Samuel M. Meierb,d and Bernhard K. Kepplera,b

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2017

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Materials and Methods

Materials

Chemicals (at least analytical grade), if not otherwise stated, were obtained from commercial

suppliers and used as received. Ubiquitin (from bovine erythrocytes), [tri(acetylacetonato)cobalt(III)]

(99.99% trace metals basis) and 3-nitrobenzyl alcohol (for mass spectrometry, ≥99.5%) were

purchased from Sigma-Aldrich, ammonium bicarbonate (BioUltra, ≥99.5%), formic acid (p.a. for

mass spectrometry) and ammonium hydroxide solution (≥25% in H2O, eluent additive for LC-MS)

from Fluka and acetic acid (99.8%, for analysis) from Acros. The lyophilized oligonucleotide

5´-dATTGGCAC-3´ (HPLC purified) was obtained from Integrated DNA Technologies (Coralville,

USA) or Microsynth (Balgach, Switzerland), and methanol and 2-propanol (both HPLC grade) from

Fisher. NaOH solution (1 M) was purchased from Agilent Technologies and inorganic standards (P,

Pt, Ru, Re; 1000 ± 3 µg/mL) from CPI. Ultrapure water was obtained from a Millipore Advantage

A10 system (18.2 MΩ, ≤5 ppb TOC, 185 UV, Darmstadt, Germany). Cisplatin, RM175 and

[(cym)RuCl(maltolato)], where cym = η6-p-cymene, were synthesized according to literature

procedures.1-3

Instrumentation

Measurements were performed on a G7100 capillary electrophoresis system (Agilent Technologies,

Waldbronn, Germany) equipped with a diode-array detector (190–400 nm). High resolution mass

spectra were acquired on an ESI-qTOF mass spectrometer (maXis 4G UHR-TOF, Bruker, Bremen,

Germany) and ICP-MS data were acquired on an Agilent 7800 or 8800 (Waldbronn, Germany).

Analyte Preparation

Stock solutions of ubiquitin (ub), DNA-oligomer (DNA, 200 µM each), and the metal complexes

cisplatin, RM175 and [(cym)RuCl(maltolato)] (1 mM each) were prepared in water. Reaction

solutions were prepared by adding the metallodrugs to the biomolecule mixture to yield final

concentrations of 50 µM for each ub/DNA and 200 µM for the respective metallodrug. They were

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incubated at 37 °C at constant shaking (400 rpm) in the dark. Aliquots were taken after 1 and 24 h of

incubation.

CZE-MS

Capillaries (fused silica, undeactivated, 75 µm, Agilent Technologies, USA) were cut to a total length

of 70 cm with a ShortixTM capillary column cutter (SGT, Singapore) to obtain a clean edge. About

5 mm of the protective polyimide outer capillary coating was removed at both ends of the capillaries

by flaming. The UV/Vis detection window was self-made 23 cm from the inlet end. The capillary was

wiped clean with isopropanol before installation. New capillaries were flushed (950 mbar)

successively with HCl (1 M, 5 min), water (1 min), NaOH (1 M, 10 min), water (1 min) and BGE

(20 min) before use. The capillary cassette temperature was 25 °C. We used NH4HCO3 (25 mM,

pH 7.9) as background electrolyte (BGE). The separation voltage was 25 kV and capillaries were

flushed with NaOH (1 M), water and BGE for 1 min each after each run. Typical instrument

parameters can be found in Table S1.

CZE–ESI-MS. A coaxial sheath-flow CE–ESI-MS Sprayer Kit (Agilent Technologies) was used for

CE hyphenation to the ESI mass spectrometer. The sheath liquid for ESI-MS experiments consisted of

isopropanol, methanol, water, formic acid and 3-nitrobenzyl alcohol (25/25/50/0.2/0.5, v/v/v/v). The

latter was added to obtain high charge states of the protein.4 A syringe pump (KDS 1000, KD

Scientific Inc., Holliston, USA) provided the sheath flow at 3 µL/min. Typical instrument parameters

can be found in Table S1. Before injection, the capillary was rinsed with BGE for 1 min (950 mbar).

Samples were hydrodynamically injected for 5 s at 50 mbar. The electropherograms in the figures

were obtained by selecting the appropriate mass-to-charge signal including an offset of ±0.05 m/z.

CZE–ESI-MS2. In-source collison-induced dissociation (ISCID) energy was increased (90 – 170 eV)

for top-down fragmentation experiments. All other parameters were similar to CZE–ESI-MS runs.

CZE–ICP-MS. A coaxial sheath-flow CE–ESI-MS Sprayer Kit (Agilent Technologies) was used for

CE hyphenation to the ICP mass spectrometer in combination with a custom-built interface. The

sheath liquid for ICP-MS experiments was 20 mM acetic acid containing 20 ppb Re as external

standard and was provided by the nebulizer pump with an uptake of 0.03 rpm or 0.1 rpm. A solution

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of [(triacetylacetonato)cobalt(III)] with a cobalt concentration of 50 mg/L was used as an internal

standard that was diluted to a final concentration of 5 mg/L cobalt in the reaction mixtures. Typical

instrument parameters can be found in Table S1 Before injection, the capillary was rinsed with BGE

for 1 min (950 mbar). Samples were hydrodynamically injected for 10 s at 50 mbar. On the Agilent

7800 phosphorous was tracked as 31P (for cisplatin and RM175) and as 47PO on the 8800 ICP-MS (for

[(cym)RuCl(maltolato)]). To lower the spectroscopic interferences for phosphorous an oxygen cell

gas flow of 30% was applied.

Data and statistical analysis

ESI mass spectra data were analysed by ESI Compass 1.3 DataAnalysis 4.0 (Bruker Daltonics). Mass

spectra of DNA and ubiquitin were deconvoluted by the maximum entropy deconvolution algorithm

with automatic data-point spacing and an instrument resolving power of 30000. Data analysis was

carried out with Microsoft Excel. The CZE–ICP-MS experiments were baseline subtracted by

subtracting the average baseline signal between 0.5–2.0 min. Peak area was calculated by using

trapezoid rule, and summation between peak valleys was obtained with the perpendicular drop

method. Each experiment was carried out independently three times and these data sets were used for

calculating standard deviations (SDs).

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Table S1 Typical instrument parameters

CE system Agilent G7100

Capillary fs capillary, i.d. 75 µm, length 70 cm

Background electrolyte 25 mM NH4HCO3, pH 7.9

Separation Voltage 25 kV

Sample Injection Hydrodyn., ICP: 50 mbar, 10s

ESI: 50 mbar, 5s

ICP-MS system Agilent 7800

RF power 1550 W

Sample depth 8 mm

Plasma gas 15 L/min

Carrier gas flow 1.05 L/min

Auxilary Gas 0.9 L/min

Monitored isotopes 31P, 59Co, 185Re, 195Pt or 102Ru

Integration time [s] 0.2 (31P), 0.1 (59Co), 0.01 (185Re), 0.1 (195Pt or 102Ru)

Measurement statistics 100 sweeps

ICP-MS system Agilent 8800

RF power 1550 W

Sample depth 8 mm

Plasma gas 15 L/min

Carrier gas flow 1.30 L/min

Monitored isotopes 47PO, 48SO, 59Co, 185Re, 195Pt or 102Ru

Integration time [s] 0.2 (47PO), 0.1 (59Co), 0.1 (185Re), 0.1 (102Ru)

Measurement statistics 100 sweeps

Cell gas flow 30% O2

ESI-MS system Bruker maXis qTOF

capillary voltage 4.5 kV

gas flow 0.4 bar

dry gas 4 L/min

dry heater 180 °C

quadrupole energy 4 eV

collision energy 8.0 eV

ion cooler transfer time 70 µs

Spectra Rate 1 Hz

ISCID Energy 0 eV or 90 – 170 eV

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Table S2 Experimental (mexp) and theoretical (mtheor) masses of the detected species during CZE–ESI-MS experiments. The mass error is given in parts per million (Δppm).

compound species mexp mtheor Δppm

Cisplatin [DNA + 3H]3+ 803.8227 803.8237 1.2

[DNA + Pt(NH3)2 + H]3+ 879.8235 879.8254 2.2

[DNA + Pt(NH3)2Cl + 2H]3+ 891.8143 891.8170 3.0

[DNA + 2Pt(NH3)2 - H]3+ 955.4902 955.4923 2.2

[DNA + Pt(NH3)2 + Pt(NH3)2Cl]3+ 967.8155 967.8190 3.6

[DNA + 2{Pt(NH3)2Cl} + H]3+ 979.8060 979.8096 3.7

[DNA + 3{Pt(NH3)2} - 3H]3+ 1031.1568 1031.1597 2.8

[DNA + 2{Pt(NH3)2} + Pt(NH3)2Cl - 2H]3+ 1043.4814 1043.4852 3.6

[ub + 8H]8+ 1071.4592 1071.4608 1.5

[ub + Pt(NH3)2 + 6H]8+ 1099.9579 1099.9613 3.1

RM175 [(bip)Ru(en) - H]+ 315.0434 315.0433 0.3

[(bip)Ru(en)Cl]+ 351.0198 351.0197 0.3

[DNA + 3H]3+ 803.8187 803.8237 6.2

[DNA + (bip)Ru(en) - H]3+ 908.4995 908.5032 4.1

[DNA + 2{(bip)Ru(en)} - H]3+ 1013.1803 1013.1823 2.0

[ub + 8H]8+ 1071.4603 1071.4608 0.5

[ub + (bip)Ru(en) + 6H]8+ 1110.7154 1110.7155 0.1

[DNA + 3{(bip)Ru(en)} - 3H]3+ 1117.5243 1117.5278 3.1

[2DNA + 4{(bip)Ru(en)} - 3H]5+ 1215.6161 1215.6176 1.2

[DNA + 3H]3+ 803.8187 803.8237 6.2

[(cym)RuCl(maltolato)] [(cym)Ru(maltolato)]+ 361.0382 361.0377 1.4

[ub + (cym)Ru + 6H]8+ 1100.7147 1100.7108 3.5

[ub + 8H]8+ 1071.5908 1071.5861 4.4

[DNA + 2{(cym)Ru} - H]3+ 959.8269 959.8239 3.1

[DNA + 3H]3+ 803.8274 803.8237 4.6

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Table S3 Experimental (mexp) and theoretical (mtheor) masses of the selected cisplatin–biomolecule adducts during CZE–ESI-MS2 experiments. Mass errors are given in parts per million (Δppm). Capital N is a nucleoside monophosphate, n is a nucleobase, HO-N is a nucleoside, and N-f is a nucleoside 3´,5´-diphosphate methylene furane.

compound species mexp mtheor Δppm

ub + Pt(NH3)2 [1MQIFa 4 + Pt - 2H]+ 685.2149 685.2132 2.5

[1MQIFb 4 + Pt - 2H]+ 713.2125 713.2081 6.2

DNA + Pt(NH3)2 [2g + Pt(NH3) - H]+ 513.0793 513.0817 4.7

[G + Pt - H]+ 523.0081 523.0090 1.7

[G + Pt(NH3) - H]+ 540.0356 540.0355 0.2

[G-f + Pt(NH3)2 - H]+ 735.0667 735.0652 2.0

[GG + Pt(NH3)]+ 869.0892 869.0881 1.3

[TG-f + Pt(NH3) - H]+ 1022.086 1022.0848 1.2

[TTG-f + Pt(NH3) - H]+ 1326.1279 1326.1309 2.3

[TTG-f + Pt(NH3)2 - H]+ 1343.1517 1343.1574 4.2

[TTGG-f + Pt(NH3) - H]+ 1655.1753 1655.1834 4.9

[TTGG-f + Pt(NH3)2 - H]+ 1672.2055 1672.2099 2.6

[HO-ATTGG-f + Pt(NH3) - H]+ 1888.2554 1888.2750 10.4

DNA + Pt(NH3)2Cl [g + PtCl]+ 381.9876 381.9823 13.9

[g + Pt(NH3)Cl]+ 399.0112 399.0089 5.8

[G-f + Pt(NH3) - H]+ 718.0389 718.0387 0.3

[G-f + Pt(NH3)]+ 755.0161 755.0152 1.2

[G-f + Pt(NH3)2Cl]+ 772.0418 772.0418 0.0

[TTG-f + Pt(NH3)2Cl]+ 1380.1333 1380.1340 0.5

[HO-TTG-f + Pt(NH3)2Cl]+ 1613.2284 1613.2254 1.9

[TTGG-f + Pt(NH3)Cl]+ 1692.1596 1692.1601 0.3

[TTGG-f + Pt(NH3)2Cl]+ 1709.1821 1709.1866 2.6

DNA + 2{Pt(NH3)2} [a + Pt(NH3) - H]+ 346.0381 346.038 0.3

[g + Pt(NH3)2 - H]+ 379.0596 379.0589 1.8

[A + Pt - H]+ 507.0153 507.014 2.6

[2g + Pt(NH3) - H]+ 513.0838 513.0817 4.1

[G + Pt - H]+ 523.0095 523.0090 1.0

[G + Pt(NH3) - H]+ 540.0403 540.0355 8.9

[GG + Pt(NH3)]+ 869.0846 869.0881 4.0

[DNA - C + 2Pt(NH3)2 -2H]2+ 1377.2163 1377.2131 2.3

[HO-ATTGGCA + 2Pt(NH3)2 - 2H]+ 1328.1954 1328.1947 0.5

[TTGG-f + Pt(NH3) - H]+ 1655.1949 1655.1834 6.9

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Table S3 Cont.

compound species mexp mtheor Δppm

DNA + 3{Pt(NH3)2} [a + Pt(NH3) - H]+ 346.0376 346.038 1.2

[g + Pt(NH3)2 - H]+ 379.0560 379.0589 7.7

[2g + Pt - H]+ 496.0576 496.0557 3.8

[A + Pt - H]+ 507.0149 507.014 1.8

[2g + Pt(NH3) - H]+ 513.0847 513.0817 5.8

[G + Pt - H]+ 523.0130 523.0090 7.6

[G + Pt(NH3) - H]+ 540.0429 540.0355 13.7

[HO-ATTGGCA + 2Pt(NH3)2 + Pt(NH3) - 4H]2+ 1433.1802 1433.1825 1.6

Table S4 Experimental (mexp) and theoretical (mtheor) masses of selected RM175–biomolecule adducts during CZE–ESI-MS2 experiments. Mass errors are given in parts per million (Δppm) Capital N is a nucleoside monophosphate, n is a nucleobase, HO-N is a nucleoside, and N-f is a nucleoside 3´,5´-disphosphate methylene furane.

compound Species mexp mtheor ∆ppm

DNA + (bip)Ru(en) [g + (bip)Ru - H]+ 406.0263 406.0241 5.4

DNA + 2{(bip)Ru(en)} [(bip)Ru(en) - H]+ 315.0488 315.0433 17.5

[(bip)Ru + H2PO4]+ 352.9588 352.9515 20.7

[c + (bip)Ru - H]+ 366.0238 366.0179 16.1

[t + (bip)Ru - H]+ 381.0236 381.0176 15.7

[a + (bip)Ru - H]+ 390.0345 390.0292 13.6

[g + (bip)Ru - H]+ 406.0279 406.0241 9.4

[AC + (bip)Ru - H]+ 875.0877 875.0896 2.2

[AC + (bip)Ru(en) - H]+ 935.1547 935.1584 4.0

[CAC + (bip)Ru - H]+ 1164.1365 1164.1363 0.2

[TTGGCAC + (bip)Ru]2+ 1215.6729 1215.6709 1.6

[CAC + (bip)Ru(en) - H]+ 1224.2172 1224.2051 9.9

[TTGGCAC + (bip)Ru(en)]2+ 1245.7034 1245.7053 1.5

[HO-ATT-f + (bip)Ru - H]+ 1274.1609 1274.1620 0.9

[DNA - c + (bip)Ru(en)]2+ 1306.7274 1306.7294 1.5

[HO-ATT-f + (bip)Ru(en) - H]+ 1333.2236 1333.2230 0.5

[DNA + (bip)Ru(en)]2+ 1362.2491 1362.2511 1.5

[DNA - c + 2(bip)Ru - en - 2H]2+ 1433.7099 1433.7137 2.7

[DNA - c + 2(bip)Ru(en) - 2H]2+ 1463.7479 1463.7481 0.1

[GCAC + (bip)Ru - H]+ 1493.1880 1493.1891 0.7

[DNA + 2(bip)Ru(en) - 2H]2+ 1519.2702 1519.2697 0.3

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Table S4 Cont.

compound Species mexp mex ∆ppm

DNA + 2{(bip)Ru(en)} [DNA + 2(bip)Ru(en) + Na - H]2+ 1530.7589 1530.7646 3.7

[GCAC + (bip)Ru(en) - H]+ 1553.2536 1553.2579 2.8

[HO-ATTG-f + (bip)Ru - H]+ 1603.2136 1603.2148 0.7

[HO-ATTG-f + (bip)Ru(en) - H]+ 1663.2836 1663.2836 0.0

[HO-ATTGG-f + (bip)Ru - H]+ 1932.2673 1932.2677 0.2

[HO-ATTGG-f + (bip)Ru(en) - H]+ 1992.3356 1992.3365 0.5

DNA + 3{(bip)Ru(en)} [(bip)Ru(en) - H]+ 315.0499 315.0433 20.9

[(bip)Ru + H2PO4]+ 352.9573 352.9515 16.4

[c + (bip)Ru - H]+ 366.0243 366.0179 17.5

[t + (bip)Ru - H]+ 381.0225 381.0176 12.9

[a + (bip)Ru - H]+ 390.0336 390.0292 11.3

[g + (bip)Ru - H]+ 406.0278 406.0241 9.1

[AC-OH + (bip)Ru - H]+ 875.0854 875.0896 4.8

[DNA - c + 3(bip)Ru(en) - 3H]3+ 975.4967 975.4959 0.8

[DNA + (bip)Ru(en)]2+ 1362.2481 1362.2511 2.2

[DNA - c + 2(bip)Ru(en) - 2H]2+ 1463.7465 1463.7481 1.1

[GCAC + (bip)Ru - H]+ 1493.1854 1493.1891 2.5

[DNA + 2(bip)Ru(en) - 2H]2+ 1519.2693 1519.2697 0.3

[GCAC + (bip)Ru(en) - H]+ 1553.2569 1553.2579 0.6

[HO-ATTG-f + (bip)Ru - H]+ 1603.2162 1603.2148 0.9

[HO-ATTG-f + (bip)Ru(en) - H]+ 1663.2844 1663.2836 0.5

[HO-ATTGG-f + (bip)Ru - H]+ 1932.2660 1932.2677 0.9

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Figure S1 Experimental and simulated mass spectra of selected fragments of the DNA + Pt(NH3)2Cl mono-adduct depicting the additional isotopic pattern of chloride.

Figure S2 Top-down mass spectrum of the ubiquitin-cisplatin mono-adduct ub + Pt(NH3)2.

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Figure S3 Plot of the relative peak areas of the 195Pt and 31P traces (n = 3) corresponding to selected species from the interaction between cisplatin and the oligonucleotide.

Figure S4 Selected fragments from the top-down analysis of a reaction mixture containing RM175 and the oligonucleotide. The metallated adducts selected for top-down analysis correspond to DNA + 3{(bip)Ru(en)} (A) and DNA + {(bip)Ru(en)} (B).

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Figure S5 Extracted ion electropherograms (EIEs) of the [(cym)RuCl(maltolato)]-biomolecule adducts by CZE-ESI-MS (A) and CZE-ICP-MS (B) after incubation with a mixture of oligonucleotide and protein (1:1) for 1 and 24 h.

Figure S6 Selected fragment from the top-down analysis of the ub + (cym)Ru adduct from the reaction identifying Met1 as the primary binding site of [(cym)RuCl(maltolato)] on ub.

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References

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Parsons, A. Parkin, G. Boyd, D. I. Jodrell and P. J. Sadler, J. Med. Chem., 2001, 44, 3616-3621.3. W. Kandioller, C. G. Hartinger, A. A. Nazarov, M. L. Kuznetsov, R. O. John, C. Bartel, M. A.

Jakupec, V. B. Arion and B. K. Keppler, Organometallics, 2009, 28, 4249-4251.4. G. Bonvin, S. Rudaz and J. Schappler, Anal. Chim. Acta, 2014, 813, 97-105.